Synthesis 2016; 48(08): 1108-1116
DOI: 10.1055/s-0035-1561220
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© Georg Thieme Verlag Stuttgart · New York

Synthesis of 5-Hydroxymethylcytidine- and 5-Hydroxymethyl­uridine-Modified RNA

Christian Riml, Ronald Micura*
  • Institute of Organic Chemistry and Center for Molecular Biosciences, CMBI, Leopold-Franzens University, Innrain 80-82, 6020 Innsbruck, Austria   Email: ronald.micura@uibk.ac.at
Further Information

Publication History

Received: 25 November 2015

Accepted after revision: 07 January 2015

Publication Date:
26 January 2016 (eFirst)

 

Abstract

We report on the syntheses of 5-hydroxymethyl-uridine [5hm(rU)] and -cytidine [5hm(rC)] phosphoramidites and their incorporation into RNA by solid-phase synthesis. Deprotection of the oligonucleotides is accomplished in a straightforward manner using standard conditions, confirming the appropriateness of the acetyl protection used for the pseudobenzylic alcohol moieties. The approach provides robust access to 5hm(rC/U)-modified RNAs that await applications in pull-down experiments to identify potential modification enzymes. They will also serve as synthetic probes for the development of high-throughput-sequencing methods in native RNAs.

1 Introduction

2 Protection Strategies Reported for the Synthesis of 5hm(dC)-Modified DNA

3 Synthesis of 5-Hydroxymethylpyrimidine-Modified RNA

3.1 Synthesis of 5hm(rC) Phosphoramidite

3.2 Synthesis of 5hm(rU) Phosphoramidite

3.3 Synthesis of 5hm(rC)- and 5hm(rU)-Modified RNA

4 Conclusions


#

Biographical Sketches

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Christian Riml was born in Zams (Austria) in 1986. He received his Magister degree in chemistry in 2012 from the Leopold-Franzens University in Innsbruck. In 2013, he was awarded a Marshall Plan Scholarship to conduct research at the University of New Orleans (USA). He returned to Innsbruck to successfully complete the teacher training program in chemistry and biology at the University of Innsbruck in 2014. He is currently pursuing his Ph.D. degree in organic chemistry under the supervision of Ronald Micura. His research focuses on modified nucleosides in the context of RNA epigenetics.

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Ronald Micura was born in 1970, and studied chemistry at the Johannes-Kepler University in Linz (Austria). He received his Ph.D. in the field of phycobilin pigments under the supervision of Karl Grubmayr in 1995. Immediately thereafter he joined the laboratory of Albert Eschenmoser at ETH Zurich (Switzerland) (1996–1997), and then moved to The Scripps Research Institute (USA) (1998), to perform postdoctoral research on alternative nucleic acids. Following his time in California, he started independent research funded by an APART-fellowship from the Austrian Academy of Science (OeAW) at the University of Linz. In 2004, he was appointed Professor of Organic Chemistry at the Leopold-Franzens University in Innsbruck (Austria). His research focuses on the chemistry, chemical biology, and biophysics of RNA.

Introduction

The nucleobase modification 5-methylcytosine (5mC) shows widespread occurrence in DNA and RNA.[1] Currently, the vast majority of efforts to understand the role of cytosine methylation, its function and its metabolism is focused on DNA because of its prominent role in epigenetics.[2] Epigenetic research seeks to describe cellular and physiological phenotypic-trait variations that are caused by external or environmental factors that switch genes on and off. DNA methylation–demethylation is such a mechanism that triggers functionally relevant changes which determine how genes are expressed without altering the underlying DNA sequence, and thus generates dynamic modulations in the transcriptional profile of a cell.[3] While the molecular basis of cytosine methylation has been well studied over the decades, and a lot is known on the enzymatic installation of a methyl group by cytosine-5 methyltransferases involving the cofactors S-adenosyl methionine (SAM) or cobalamin (Cbl),[4] [5] the demethylation of 5mC has been explored only recently in detail.[6,7] One of the main demethylation pathways of 5mC suggests 5-hydroxymethylcytosine (5hmC) to be the first degradation product of enzymatic oxidation by ketoglutarate-dependent hydroxylases of the ten-eleven-translocation (TET) family of proteins.[8]

In RNA, 5mC is also prevalent,[9] [10] [11] [12] however, little is known if this modification is dynamic and plays a role in post-transcriptional regulation.[13] In the emerging field of RNA epigenetics,[14] the question if a similar oxidative reversal pathway may also work for the 5mC modification in RNA has challenged several laboratories, but to the best of our knowledge, the native enzymes catalyzing such reactions in RNA have not yet been identified.[15]

The present work aims at generating synthetic RNA probes that contain 5-hydroxymethyluridine [5hm(rU)] and/or 5-hydroxymethylcytidine [5hm(rC)] modifications. Synthetic access to site-specifically modified 5hm(rC/U)-containing RNA provides the foundation for pull-down experiments which are needed to identify proteins that might specifically recognize and process these modifications in RNA.[16] [17] Moreover, efficient access to 5hm(rU)- and/or 5hm(rC)-containing probes will also be invaluable to develop high-throughput-sequencing methods to identify these modifications in native RNA.[18–20]


# 2

Protection Strategies Reported for the Synthesis of 5hm(dC)-Modified DNA

On first glance, 5-hydroxymethylcytosine-modified nucleic acids appear to be easily accessible by chemical synthesis. However, this modification has been reported to be rather challenging for solid-phase approaches because of the observed SN reactivity that originates from its pseudobenzylic character.[21] [22] With respect to oligodeoxynucleotide synthesis, several distinct protecting groups have been described in the literature for the 5-hydroxymethyl group of 5-hydroxymethyl-2'-deoxycytidine [5hm(dC)] (Figure [1]). Among these are the 2-cyanoethyl,[23] [24] acetyl,[25] and tert-butyldimethylsilyl (TBDMS) groups.[26] Additionally, the simultaneous protection of the 5-hydroxymethyl and the C4-NH2 group as a cyclic carbamate has been described.[27] [28] Although these approaches have their merits, they are also not entirely satisfying. For instance, post-synthetic removal of the cyanoethyl protecting group was reported to be troublesome with respect to completeness.[23] Also, not fully convincing was that carbamate deprotection required NaOH in methanol/water instead of the standard DNA deprotection (ammonolysis) to avoid aminocarbonyl-N 4 and 5-aminomethylcytosine byproducts.[27] When 5hm(dC) was protected with 5-CH2-OTBDMS together with N 4-benzoyl, the deprotection procedure came closest to standard DNA deprotection, however, extended reaction times and elevated temperatures had to be applied in order to demask the 5-CH2OH group completely and simultaneously with the base-labile protecting groups in concentrated aqueous ammonium hydroxide solution.[26]

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Figure 1 Different protection patterns of 5-hydroxymethyl-2′-deoxycytidine phosphoramidites for the solid-phase synthesis of 5hm(dC)-containing DNA that have been reported in the literature

# 3

Synthesis of 5-Hydroxymethylpyrimidine-Modified RNA

To the best of our knowledge, the synthesis of 5-hydroxymethylcytidine [(5hm(rC)] modified RNA has not yet been accomplished. To achieve this aim, we considered acetylation of both the 5-hydroxymethyl and the N 4 exo­cyclic amino groups as a promising protection pattern for building blocks that can be utilized in RNA solid-phase synthesis. At first sight, such a concept may be surprising since it was reported earlier that the incorporation of an O-acetylated and N4-benzoylated 5hm(dC) building block into DNA gave undesired benzamide byproducts that originated from nucleophilic displacement of the acetate moiety and migration of the benzoyl group during oligonucleotide deprotection with ammonium hydroxide.[25] However, in contrast to synthetic DNA, RNA is routinely deprotected by applying more nucleophilic conditions of methylamine in ethanol/water, or alternatively, of methylamine and ammonia in water. Under these conditions, RNA is deprotected very fast and cleanly; importantly, acetyl (instead of benzoyl) protection has to be used for the exocyclic N 4 amino group to avoid transamination at C4 by methylamine.[29] [30] Considering all the above arguments, we set out to synthesize and evaluate the bisacetylated 5hm(rC) building block 9 for RNA solid-phase synthesis.

3.1

Synthesis of 5hm(rC) Phosphoramidite

We conceived the synthesis of 5hm(rC) phosphoramidite 9 from nucleoside 1 [5hm(rU)] as the starting point for two reasons (Scheme [1]). First, access to 5hm(rU) 1 (or derivatives thereof) has been described,[31] [32] [33] [34] [35] [36] and second, we considered it desirable to introduce a divergent synthetic pathway for phosphoramidites of both nucleosides 5hm(rU) and 5hm(rC). Hence, a uracil-into-cytosine conversion was envisaged, a transformation that is well established for various pyrimidine nucleoside modifications, with a wide range of conditions accepted.[37–39]

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Scheme 1 Synthesis of 5hm(rC) phosphoramidite 9. Reaction conditions: (a) TFA (0.0002 equiv), AcOH, reflux, 40 min, 62%; (b) DMT-Cl (1.2 equiv), DMAP (0.025 equiv), pyridine, 5 h, r.t., 65%; (c) TBDMSCl (3.0 equiv), imidazole (6.0 equiv), DMF, r.t., 5 h, 80%; (d) 2,4,6-triisopropylbenzenesulfonyl chloride (1.5 equiv), Et3N (10.0 equiv), DMAP (0.12 equiv), CH2Cl2, r.t., 1 h, no purification; (e) aq NH3 (32%), THF, r.t., 3 h, 44% over 2 steps; (f) Ac2O (2.5 equiv), pyridine, 0 °C to r.t., 2 h, 76%; (g) 1 M TBAF/0.5 M AcOH, THF, r.t., 3 h, 75%; (h) AgNO3 (1.6 equiv), TBDMSCl (1.6 equiv), pyridine (3.5 equiv), THF, r.t., 4 h, 43%; (i) CEP-Cl (2.0 equiv), DIPEA (4.0 equiv), 1-methylimidazole (0.5 equiv), CH2Cl2, r.t., 5 h, 79%.

We started the synthesis with selective substitution of the hydroxy group of 5-hydroxy-methylated uridine 5hm(rU) 1 by acetic acid using a catalytic amount of trifluoroacetic acid (Scheme [1]).[31] It should be pointed out that reaction times longer than 40 minutes resulted in additional acetylation of the ribose hydroxy groups. The monoacetylated derivative 2 was then transformed into the dimethoxytritylated compound 3, followed by protection of the 2′- and 3′-hydroxy groups as TBDMS silyl ethers to yield nucleoside 4. Next, reaction of 4 with 2,4,6-triisopropylbenzenesulfonyl chloride in the presence of triethylamine and DMAP in dichloromethane resulted in regioselective O 4-trisylation. After work-up, the trisylated derivative 4a could be used without further purification for the conversion into 5 upon treatment with aqueous ammonium hydroxide in THF. Acetylation of the amino function was achieved with acetic anhydride in pyridine to provide 6, followed by cleavage of the 2′- and 3′-OTBDMS groups with TBAF (1 M) and acetic acid (0.5 M) in THF to give 7. Selective silylation with TBDMSCl using silver nitrate according to Ogilvie[40] provided the 2′-OTBDMS-protected derivative 8 in 43% yield. To increase the overall yield, the undesired 3′-O-silylated regioisomer was treated with triethylamine in methanol, with equilibration again generating a favorable distribution between the 2′ and 3′ regioisomers. Finally, phosphitylation was executed with 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite (CEP-Cl) in the presence of N,N-diisopropylethylamine in CH2Cl2. Starting with compound 1, our route provides product 9 in a 3% overall yield in nine steps and with eight chromatographic purifications; in total, 0.5 gram of 9 was obtained in the course of this study.

Concerning the overall synthetic strategy for phosphoramidite 9, we mention that we did not consider 2′-O-tomylation of intermediate 3, followed by 3′-O-acetylation and U-to-C transformation.[37] [38] [39] The reason being that such a path would require selective cleavage of the temporary 3′-OAc group in the presence of the 5-AcOCH2 and NHAc moieties, prior to transformation into phosphoramidite 9. Another reason was that the yields for introducing the 2′-O-[(triisopropylsilyl)oxy]methyl (TOM) group on uridines are generally lower compared to the introduction of the 2′-OTBDMS group.[40] [41] [42]

Furthermore, we found that attempts to shorten the reaction sequence leading from compound 6 directly to 8 (by deprotection of one of the 2′-O/3′-OTBDMS groups and subsequent equilibration) suffered from good reproducibility, so that we recommend the here-elaborated path for reasons of robustness.


# 3.2

Synthesis of 5hm(rU) Phosphoramidite

As mentioned above, a divergent synthetic route was designed to allow access also to 5hm(rU) phosphoramidite building blocks. Starting from derivative 3 (Scheme [2]), silylation with TBDMSCl using silver nitrate according to Ogilvie[40] yielded the 2′-OTBDMS-protected derivative 10 with remarkable regioselectivity over the 3′-O-silylated product (5:1). Phosphitylation was executed with CEP-Cl in the presence of N,N-diisopropylethylamine in THF. Starting from 5hm(rU) 1, the corresponding phosphoramidite 11 was isolated in an overall yield of 14% over four steps with four chromatographic purifications; in total, 0.6 gram of 11 was obtained in the course of this study.

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Scheme 2 Synthesis of 5hm(rU) phosphoramidite 11. Reaction conditions: (a) AgNO3 (1.6 equiv), TBDMSCl (1.6 equiv), pyridine (3.5 equiv), THF, r.t., 4 h, 44%; (b) CEP-Cl (2.0 equiv), DIPEA (4.0 equiv), 1-methylimidazole (0.5 equiv), CH2Cl2, r.t., 5 h, 80%.

# 3.3

Synthesis of 5hm(rC)- and 5hm(rU)-Modified RNA

The solid-phase synthesis of RNA with site-specific 5hm(rC/U) modifications was performed following the TOM approach.[41] [43] [44] Coupling yields of the novel building block were higher than 98% according to the trityl assay. Cleavage of the synthetic RNA strands from the solid support and their deprotections were performed using methylamine in ethanol/water, or alternatively methylamine/ammonia in water followed by treatment with TBAF in THF. Salts were removed by size-exclusion chromatography on a Sephadex G25 column. In general, the crude products gave a major product peak in anion-exchange (AE) HPLC analysis. RNA oligomers were purified by AE chromatography under strong denaturing conditions (6 M urea, 80 °C) (Figure [2]). The molecular weights of the purified RNAs were confirmed by liquid chromatography–electrospray ionization (LC–ESI) mass spectrometry (MS). The synthesized RNAs containing 5hm(rC) and 5hm(rU) modifications are listed in Table [1].

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Figure 2 Characterization of 5hm(rC)- and 5hm(rU)-modified RNA. Anion-exchange HPLC traces (top) of: (A) 8 nt RNA, (B) 15 nt RNA, and (C) 15 nt RNA, and the corresponding LC–ESI mass spectra (bottom). HPLC conditions: Dionex DNAPac column (4 × 250 mm), 80 °C, 1 mL min–1, 0–60% buffer B in buffer A within 45 min; buffer A: Tris-HCl (25 mM), urea (6 M), pH 8.0; buffer B: Tris-HCl (25 mM), urea (6 M), NaClO4 (0.5 M), pH 8.0. See the experimental for LC–ESI MS conditions

Table 1 Selection of Synthesized RNAs with 5hm(rC), 5hm(rU) and 5m(rC) Modificationsa

Sequence (5′→3′)

Amount (nmol)

MWcalcd (amu)

MWobsd (amu)

GU( hmC)ACC (6 nt)

280

1864.2

1863.9

GG( hmC)UAG( hmC)C (8 nt)

 43

2584.6

2584.4

GAAGGGCAAC( hmC)UUCG (15 nt)

215

4844.0

4844.0

GCGAACCUG( hmC)GGGUUCG (17 nt)

 89

5487.3

5487.2

GU( hmU)ACC (6 nt)

354

1865.2

1865.0

GUC( hmU)AGAC (8 nt)

326

2539.6

2539.6

GCAAGGCAAC( hmU)UGCG (15 nt)

161

4844.0

4843.9

AUCUGCUUG( mC)( hmC)( mC)AUCGGGG( hmC)CGCGGAU (27 nt)

 70

8704.2

8705.2

a ( hmC) = 5-Hydroxymethylcytidine, ( hmU) = 5-hydroxymethyluridine, ( mC) = 5-methylcytidine.

It is noteworthy that minor amounts of byproducts (<15% of the crude deprotected material) that originated from substitution reactions at the 5-acetyloxymethyl group by methylamine during RNA deprotection were found, as analyzed by LC–ESI mass spectrometry. However, these byproducts were easily separated from the desired product during anion-exchange and/or reversed-phase chromato­graphy. In this context, deprotection using equal volumes of aqueous methylamine (40%) and saturated aqueous NH3 solutions appeared slightly favorable over ‘methylamine alone’ conditions.


#
# 4

Conclusions

We have elaborated the syntheses of 5-acetyloxy-methylated cytidine and uridine phosphoramidites and the incorporation of these building blocks into RNA by solid-phase synthesis. Importantly, we have demonstrated that subsequent deprotection of the oligoribonucleotides is straightforward using aqueous methylamine/ammonia solutions. Only very minor amounts of side products that resulted from the SN reactivity at the pseudobenzylic position of the 5-hydroxymethyl residues were observed. Consequently, this approach delivers high-quality 5hm(rC/U)-modified RNAs as valuable probes in the search for related modification enzymes and for the development of high-throughput-sequencing methods in the emerging field of RNA epigenetics.


#

All reactions were carried out under an argon atmosphere. Chemical reagents and solvents were purchased from commercial suppliers and were used without further purification. Organic solvents for reactions were dried overnight over freshly activated molecular sieves (3 Å). Analytical thin-layer chromatography (TLC) was carried out on Macherey-Nagel Polygram SIL G/UV254 plates. Flash column chromatography was carried out on Sigma Aldrich silica gel 60 (70−230 mesh). 1H, 13C and 31P NMR spectra were recorded on a Bruker DRX 300 MHz instrument. The chemical shifts are referenced to the residual proton signal of the deuterated solvents: CDCl3 (7.26 ppm), DMSO-d 6 (2.49 ppm) for 1H NMR spectra; CDCl3 (77.0 ppm) or DMSO-d 6 (39.5 ppm) for 13C NMR spectra. 1H and 13C NMR assignments were based on COSY and HSQC experiments. MS experiments were performed on a Finnigan LCQ Advantage MAX ion trap instrument.


#

5-Acetyloxymethyluridine (2)

5-Hydroxymethyluridine (1) (obtained by various routes as described in references 31–33) (3.3 g, 12 mmol) was suspended in glacial AcOH (100 mL). TFA (0.2 mL) was added and the mixture was refluxed for 40 min. AcOH was removed under reduced pressure. The crude product was purified by column chromatography on silica gel (CH2Cl2/ MeOH, 95:5 to 90:10). Although the product 2 can be contaminated with up to 10% of the 5′-O-acetylated regioisomer, it should be used without further purification in the next step. This byproduct becomes easily separated from compound 3 by column chromatography (see below).

Yield: 2.4 g, 7.5 mmol (62%); white powder; Rf  = 0.7 (CH2Cl2/MeOH, 75:25).

1H NMR (300 MHz, DMSO-d 6): δ = 2.00 (s, 3 H, H 3C-CO), 3.57 (m, 1 H, H(1)-C(5′)), 3.63 (m, 1 H, H(2)-C(5′)), 3.85 (m, 1 H, H-C(4′)), 3.94–4.07 (m, 2 H, H-C(3′), H-C(2′)), 4.68 (s, 2 H, H 2C-C(5)), 5.08–5.10 (m, 2 H, HO-C(5′), HO-C(3′)), 5.38 (d, J = 5.5 Hz, 1 H, HO-C(2′)), 5.77 (d, J = 5.1 Hz, 1 H, H-C(1′)), 8.08 (s, 1 H, H-C(6)), 11.49 (s, 1 H, H-N(3)).

13C NMR (75 MHz, DMSO-d 6): δ = 21.24 (COCH3), 59.57 (CH2OH), 61.34 (C(5′)), 70.33 (C(3′)), 74.22 (C(2′)), 85.46 (C(4′)), 88.45 (C(1′)), 108.86 (C(5)), 141.31 (C(6)), 151.06 (C(2)), 163.01 (C(4)), 170.83 (COCH3).

MS (ESI): m/z [M + H]+ calcd for C12H16N2O8: 317.10; found: 316.93.


#

5-Acetyloxymethyl-5′-O-(4,4′-dimethoxytrityl)uridine (3)

5-Acetyloxymethyluridine (2) (2.4 g, 7.5 mmol) and DMAP (20 mg, 0.18 mmol) were dissolved in dry pyridine (10 mL). 4,4′-Dimethoxytrityl chloride (DMT-Cl) (3 g, 9 mmol) was added in two portions. The reaction was stirred for 5 h at r.t., and the progress monitored by TLC. The pyridine was removed under reduced pressure, the residue dissolved in CH2Cl2 and extracted with 5% citric acid (2 ×), sat. aq NaHCO solution and sat. aq NaCl solution. The organic phase was dried over anhydrous Na2SO4 and coevaporated twice with toluene and with CH2Cl2. The crude product was purified by column chromatography on silica gel (CH2Cl2/MeOH, 98:2–96:4).

Yield: 3 g, 4.8 mmol (65%); white foam; Rf  = 0.45 (CH2Cl2/MeOH, 95:5).

1H NMR (300 MHz, DMSO-d 6): δ = 1.86 (s, 3 H, H 3C-CO), 3.15–3.28 (m, 2 H, H 2-C(5′)), 3.73 (s, 6 H, H 3C-O), 3.97 (m, 1 H, H-C(4′)), 4.07 (m, 1 H, H-C(3′)), 4.16 (m, 1 H, H-C(2′)), 4.29 (m, 2 H, H 2C-C(5)), 5.17 (d, J = 5.7 Hz, 1 H, HO-C(3′)), 5.50 (d, J = 5.4 Hz, 1 H, HO-C(2′)), 5.79 (d, J = 4.6 Hz, 1 H, H-C(1′)), 6.86–6.89 (m, 4 H, H-C(Ar)), 7.22–7.39 (m, 9 H, H-C(Ar)), 7.79 (s, 1 H, H-C(6)), 11.57 (s, 1 H, H-N(3)).

13C NMR (75 MHz, DMSO-d 6): δ = 21.02 (COCH3), 55.60 (2 × OCH3), 59.28 (CH2OH), 64.11 (C(5′)), 70.55 (C(3′)), 73.86 (C(2′)), 83.45 (C(4′)), 86.42 (t-C(DMT)), 89.28 (C(1′)), 108.96 (C(5)), 113.84 (C(Ar)), 127.38 (C(Ar)), 128.30 (C(Ar)), 128.49 (C(Ar)), 130.30 (C(Ar)), 135.85 (C(Ar)), 141.53 (C(6)), 145.16 (C(Ar)), 150.95 (C(2)), 158.74 (C-OCH3(Ar)), 162.98 (C(4)), 170.55 (COCH3).

MS (ESI): m/z [M + Na]+ calcd for C33H34N2O10: 641.20; found: 641.15.


#

5-Acetyloxymethyl-5′-O-(4,4′-dimethoxytrityl)-2′,3′-di-O-tert-butyldimethylsilyluridine (4)

Compound 3 (3 g, 4.8 mmol) was dissolved in DMF (15 mL). Imidazole (1.95 g, 28.8 mmol) and TBDMSCl (2.16 g, 14.4 mmol) were added consecutively, and the solution was stirred at r.t. for 5 h. The excess of silyl reagent was quenched by slow addition of MeOH (10 mL). H2O (500 mL) was added and the mixture was extracted with EtOAc (3 × 200 mL). The combined organic layers were dried over Na2SO4 and the solvents evaporated. The crude product was purified by column chromatography on silica gel (hexane/EtOAc, 2:1).

Yield: 3.4 g, 4.1 mmol (80%); white solid; Rf  = 0.6 (hexane/EtOAc, 50:50).

1H NMR (300 MHz, DMSO-d 6): δ = –0.08 (s, 3 H, H 3C-Si), 0.00 (s, 3 H, H 3C-Si), 0.03 (s, 6 H, H 3C-Si), 0.74 (s, 9 H, (H 3C)3-C), 0.84 (s, 9 H, (H 3C)3-C), 1.83 (s, 3 H, H 3C-CO), 3.23–3.35 (m, 2 H, H 2-C(5′)), 3.73 (s, 6 H, H 3C-O), 3.99 (m, 1 H, H-C(4′)), 4.07 (m, 1 H, H-C(3′)), 4.16 (m, 1 H, H-C(2′)), 4.29–4.42 (m, 2 H, H 2C-C(5)), 5.77 (d, J = 4.3 Hz, 1 H, H-C(1′)), 6.86–6.89 (m, 4 H, H-C(Ar)), 7.22–7.39 (m, 9 H, H-C(Ar)), 7.82 (s, 1 H, H-C(6)), 11.62 (s, 1 H, H-N(3)).

13C NMR (75 MHz, DMSO-d 6): δ = –4.30 (4 × CH3-Si), 18.16 (2 × C(CH3)3), 21.02 (COCH3), 26.19 (6 × CH3-C-Si), 55.63 (2 × OCH3), 59.13 (CH2OH), 63.61 (C(5′)), 72.16 (C(3′)), 75.09 (C(2′)), 83.89 (C(4′)), 86.77 (t-C(DMT)), 88.90 (C(1′)), 109.13 (C(5)), 113.84 (C(Ar)), 127.49 (C(Ar)), 128.30 (C(Ar)), 128.49 (C(Ar)), 130.30 (C(Ar)), 135.60 (C(Ar)), 140.93 (C(6)), 145.02 (C(Ar)), 150.84 (C(2)), 158.86 (C-OCH3(Ar)), 162.93 (C(4)), 170.55 (COCH3).

MS (ESI): m/z [M + Na]+ calcd for C45H62N2O10Si2: 869.38; found: 869.40.


#

5-Acetyloxymethyl-5′-O-(4,4′-dimethoxytrityl)-2′,3′-di-O-tert-butyldimethylsilyl-O 4-(2,4,6-triisopropylbenzenesulfonyl)uridine (4a)

To a solution of compound 4 (3.4 g, 4.1 mmol) in dry CH2Cl2 (20 mL) was added DMAP (40 mg, 0.36 mmol) and Et3N (5.6 mL, 41 mmol). 2,4,6-Triisopropylbenzenesulfonyl chloride (1.8 g, 6.15 mmol) was added slowly, and the solution was stirred for 1 h at r.t. The reaction mixture was diluted with CH2Cl2, washed with sat. NaHCO3 solution, and the organic phase was dried over Na2SO4, filtered, and evaporated. The crude product 4a was obtained as a yellow foam and was used for the next step without further purification. An analytical sample of 4a was purified by column chromatography on silica gel (hexane/ EtOAc, 90:10–85:15).

Rf  = 0.5 (hexane/EtOAc, 75:25).

1H NMR (300 MHz, DMSO-d 6): δ = –0.14 (s, 3 H, H 3C-Si), –0.07 (s, 3 H, H 3C-Si), –0.04 (s, 3 H, H 3C-Si), –0.03 (s, 3 H, H 3C-Si), 0.67 (s, 9 H, (H 3C)3-C), 0.77 (s, 9 H, (H 3C)3-C), 1.20–1.25 (m, 18 H, (H 3C)2CH), 1.85 (s, 3 H, H 3C-CO), 2.95 (m, 1 H, (H3C)2CH)), 3.24–3.44 (m, 2 H, H 2-C(5′)), 3.72 (s, 6 H, H 3C-O), 3.95 (m, 1 H, H-C(4′)), 4.07 (m, 1 H, H-C(3′)), 4.16–4.42 (5 H, H-C(2′), 2 × (H3C)2CH, H 2C-C(5)), 5.66 (d, J = 2.9 Hz, 1 H, H-C(1′)), 6.86–6.89 (m, 4 H, H-C(Ar)), 7.22–7.39 (m, 11 H, H-C(Ar)), 8.33 (s, 1 H, H-C(6)).


#

5-Acetyloxymethyl-5′-O-(4,4′-dimethoxytrityl)-2′,3′-di-O-tert-butyldimethylsilylcytidine (5)

The crude compound 4a (4.1 mmol) was dissolved in THF (50 mL) and treated with aq NH3 (32%, 70 mL). The solution was stirred at r.t. for 3 h. The solvents were evaporated, and the mixture was diluted with CH2Cl2 and washed with H2O. The organic phase was dried over Na2SO and the solvents evaporated. The crude product was purified by column chromatography on silica gel (CH2Cl2/MeOH, 100:0–98:2).

Yield: 1.5 g, 1.8 mmol (44% over 2 steps); white foam; Rf  = 0.5 (CH2Cl2/ MeOH, 95:5).

1H NMR (300 MHz, DMSO-d 6): δ = –0.10 (s, 3 H, H 3C-Si), –0.02 (s, 3 H, H 3C-Si), 0.02 (s, 3 H, H 3C-Si), 0.04 (s, 3 H, H 3C-Si), 0.74 (s, 9 H, (H 3C)3-C), 0.84 (s, 9 H, (H 3C)3-C), 1.90 (s, 3 H, H 3C-CO), 3.20–3.35 (m, 2 H, H 2-C(5′)), 3.73 (s, 6 H, H 3C-O), 3.97 (m, 2 H, H-C(4′), H-C(3′)), 4.22 (1 H, H-C(2′)), 4.34–4.44 (m, 2 H, H 2C-C(5)), 5.81 (d, J = 4.0 Hz, 1 H, H-C(1′)), 6.86–6.89 (m, 4 H, H-C(Ar)), 7.05 (s, 1 H, H(a)-N(4)), 7.22–7.41 (m, 9 H, H-C(Ar)), 7.52 (s, 1 H, H(b)-N(4)), 7.76 (s, 1 H, H-C(6)).

13C NMR (75 MHz, DMSO-d 6): δ = –4.30 (4 × CH3-Si), 18.20 (2 × C(CH3)3), 21.27 (COCH3), 26.20 (6 × CH3-C-Si), 55.61 (2 × OCH3), 60.83 (CH2OH), 63.74 (C(5′)), 72.16 (C(3′)), 75.76 (C(2′)), 83.24 (C(4′)), 86.65 (t-C(DMT)), 89.40 (C(1′)), 101.26 (C(5)), 113.80 (C(Ar)), 127.44 (C(Ar)), 128.30 (C(Ar)), 128.49 (C(Ar)), 130.30 (C(Ar)), 135.77 (C(Ar)), 142.83 (C(6)), 145.05 (C(Ar)), 155.27 (C(2)), 158.86 (C-OCH3(Ar)), 164.78 (C(4)), 170.68 (COCH3).

MS (ESI): m/z [M + Na]+ calcd for C45H63N3O9Si2: 868.40; found: 868.39.


#

N 4-Acetyl-5-acetyloxymethyl-5′-O-(4,4′-dimethoxytrityl)-2′,3′-di-O-tert-butyldimethylsilylcytidine (6)

A solution of compound 5 (1.5 g, 1.8 mmol) in dry pyridine (30 mL) was cooled to 0 °C under argon, treated with Ac2O (425 μL, 4.5 mmol), allowed to warm to r.t. and stirred for 2 h. The reaction was quenched by the addition of MeOH (2 mL), and the solvents were evaporated. The oily residue was diluted with CH2Cl2, and washed with 5% citric acid, H2O and sat. NaHCO3 solution. The organic phase was dried over Na2SO4 and the solvents were evaporated. The crude product was purified by column chromatography on silica gel (CH2Cl2/MeOH, 99.5:0.5–98:2).

Yield: 1.2 g, 1.36 mmol (76%); white foam; Rf  = 0.75 (CH2Cl2/MeOH, 95:5).

1H NMR (300 MHz, DMSO-d 6): δ = –0.12 (s, 3 H, H 3C-Si), –0.03 (s, 3 H, H 3C-Si), 0.05 (s, 3 H, H 3C-Si), 0.10 (s, 3 H, H 3C-Si), 0.68 (s, 9 H, (H 3C)3-C), 0.86 (s, 9 H, (H 3C)3-C), 1.81 (s, 3 H, H 3C-CO), 2.25 (s, 3 H, H 3C-CO-N(4)), 3.20–3.35 (m, 2 H, H 2-C(5′)), 3.73 (s, 6 H, H 3C-O), 3.99 (m, 2 H, H-C(4′)), 4.09 (m, 1 H, H-C(3′)), 4.30 (1 H, H-C(2′)), 4.38–4.61 (m, 2 H, H 2C-C(5)), 5.70 (d, J = 2.4 Hz, 1 H, H-C(1′)), 6.86–6.89 (m, 4 H, H-C(Ar)), 7.25–7.41 (m, 9 H, H-C(Ar)), 8.13 (s, 1 H, H-C(6)), 10.08 (s, 1 H, H-N(4)).

13C NMR (75 MHz, DMSO-d 6): δ = –4.80 to –3.56 (4 × CH3-Si), 18.20 (2 × C(CH3)3), 21.08 (COCH3), 25.49 (NCOCH3), 26.20 (6 × CH3-C-Si), 55.61 (2 × OCH3), 60.55 (CH2OH), 63.22 (C(5′)), 71.19 (C(3′)), 75.61 (C(2′)), 82.91 (C(4′)), 86.58 (t-C(DMT)), 91.53 (C(1′)), 105.24 (C(5)), 113.80 (C(Ar)), 127.44 (C(Ar)), 128.30 (C(Ar)), 128.49 (C(Ar)), 130.30 (C(Ar)), 135.77 (C(Ar)), 144.92 (C(6)), 145.95 (C(Ar)), 154.35 (C(2)), 158.87 (C-OCH3(Ar)), 161.77 (C(4)), 170.45 (COCH3), 171.22 (NCOCH3).

MS (ESI): m/z [M + Na]+ calcd for C47H65N3O10Si2: 910.41; found: 910.45.


#

N 4-Acetyl-5-acetyloxymethyl-5′-O-(4,4′-dimethoxytrityl)cytidine (7)

Compound 6 (1.2 g, 1.36 mmol) was treated with a solution (6 mL) of 1 M TBAF and 0.5 M AcOH in THF. The reaction mixture was stirred at r.t. for 3 h. The solvents were evaporated and the residue was coevaporated twice with CH2Cl2. The crude product was purified by column chromatography on silica gel (CH2Cl2/MeOH, 99:1–96:4).

Yield: 672 mg, 1.02 mmol (75%); white foam; Rf  = 0.45 (CH2Cl2/MeOH, 95:5).

1H NMR (300 MHz, DMSO-d 6): δ = 1.87 (s, 3 H, H 3C-CO), 2.24 (s, 3 H, H 3C-CO-N(4)), 3.25–3.32 (m, 2 H, H 2-C(5′)), 3.73 (s, 6 H, H 3C-O), 4.08 (m, 3 H, H-C(4′), H-C(3′), H-C(2′)), 4.38–4.61 (m, 2 H, H 2C-C(5)), 5.13 (d, J = 3.5 Hz, 1 H, HO-C(3′)), 5.60 (d, J = 4.5 Hz, 1 H, HO-C(2′)), 5.78 (d, J = 1.9 Hz, 1 H, H-C(1′)), 6.86–6.89 (m, 4 H, H-C(Ar)), 7.25–7.41 (m, 9 H, H-C(Ar)), 8.08 (s, 1 H, H-C(6)), 10.04 (s, 1 H, H-N(4)).

13C NMR (75 MHz, DMSO-d 6): δ = 21.12 (COCH3), 25.54 (NCOCH3), 55.60 (2 × OCH3), 60.63 (CH2OH), 63.56 (C(5′)), 69.91 (C(3′)), 74.60 (C(2′)), 83.00 (C(4′)), 86.34 (t-C(DMT)), 91.59 (C(1′)), 105.20 (C(5)), 113.85 (C(Ar)), 127.37 (C(Ar)), 128.33 (C(Ar)), 128.50 (C(Ar)), 130.30 (C(Ar)), 135.87 (C(Ar)), 136.11 (C(Ar)), 146.22 (C(6)), 145.16 (C(Ar)), 154.33 (C(2)), 158.74 (C-OCH3(Ar)), 161.79 (C(4)), 170.58 (COCH3), 171.22 (NCOCH3).

MS (ESI): m/z [M + H]+ calcd for C35H37N3O10: 660.26; found: 660.24.


#

N 4-Acetyl-5-acetyloxymethyl-5′-O-(4,4′-dimethoxytrityl)-2′-O-tert-butyldimethylsilylcytidine (8)

Compound 7 (672 mg, 1.02 mmol) was dissolved in dry THF (12 mL) and dry pyridine (300 μL) was added, followed by AgNO3 (270 mg, 1.6 mmol). The suspension was stirred for 30 min, then TBDMSCl (250 mg, 1.6 mmol) was added. Stirring was continued in the dark for 4 h until the starting material had been consumed as analyzed by TLC. The reaction mixture was filtered through Celite and the Celite bed washed with CH2Cl2 (100 mL). The combined filtrates were evaporated, the residue redissolved in CH2Cl2, and then washed with aq 5% citric acid (2 ×) and sat. NaHCO3 solution. The organic phase was dried over Na2SO4, filtered, and evaporated. The crude product was purified by column chromatography on silica gel (hexane/EtOAc, 60:40–25:75).

Yield: 340 mg, 0.44 mmol (43%); white foam; Rf  = 0.5 (hexane/EtOAc, 25:75).

1H NMR (300 MHz, DMSO-d 6): δ = 0.05 (s, 3 H, H 3C-Si), 0.07 (s, 3 H, H 3C-Si), 0.85 (s, 9 H, (H 3C)3-C), 1.84 (s, 3 H, H 3C-CO), 2.22 (s, 3 H, H 3C-CO-N(4)), 3.25–3.32 (m, 2 H, H 2-C(5′)), 3.71 (s, 6 H, H 3C-O), 4.05 (m, 2 H, H-C(4′), H-C(3′)), 4.17 (m, 1 H, H-C(2′)), 4.29–4.52 (m, 2 H, H 2C-C(5)), 5.09 (d, J = 5.5 Hz, 1 H, HO-C(3′)), 5.71 (br, 1 H, H-C(1′)), 6.86–6.89 (m, 4 H, H-C(Ar)), 7.25–7.41 (m, 9 H, H-C(Ar)), 8.04 (s, 1 H, H-C(6)), 10.02 (s, 1 H, H-N(4)). The assignments of 2′-OTBDMS (vs 3′-OTBDMS) regioisomers were based on 1H, 1H DQF COSY NMR experiments, unequivocally revealing the correlation between the H-C(3′) and HO-C(3′) (vs H-C(2′) and HO-C(2′)) signals.

13C NMR (75 MHz, DMSO-d 6): δ = –4.17, –4.36 (2 × CH3-Si), 18.57 (C(CH3)3), 21.12 (COCH3), 25.50 (NCOCH3), 26.33 (3 × CH3-C-Si), 55.61 (2 × OCH3), 60.63 (CH2OH), 63.21 (C(5′)), 69.49 (C(3′)), 76.75 (C(2′)), 82.78 (C(4′)), 86.36 (t-C(DMT)), 91.52 (C(1′)), 105.17 (C(5)), 113.85 (C(Ar)), 127.40 (C(Ar)), 128.31 (C(Ar)), 128.50 (C(Ar)), 130.30 (C(Ar)), 135.78 (C(Ar)), 136.02 (C(Ar)), 145.16 (C(6)), 145.80 (C(Ar)), 154.25 (C(2)), 158.75 (C-OCH3(Ar)), 161.82 (C(4)), 170.52 (COCH3), 171.23 (NCOCH3).

MS (ESI): m/z [M + H]+ calcd C41H51N3O10Si: 774.95; found: 774.32.


#

N 4-Acetyl-5-acetyloxymethyl-5′-O-(4,4′-dimethoxytrityl)-2′-O-tert-butyldimethylsilylcytidine 3′-O-(2-Cyanoethyl-N,N-diisopropylphosphoramidite) (9)

Compound 8 (340 mg, 0.44 mmol) was dissolved in dry CH2Cl2 (6 mL) under an argon atm. Next, DIPEA (312 μL, 1.8 mmol), 1-methylimidazole (18 μL, 0.22 mmol), and 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (195 μL, 0.88 mmol) were added to the solution using syringes. The reaction was monitored by TLC and the mixture stirred for 5 h at r.t. Then, CH2Cl2 was added and the organic phase extracted with sat. aq NaHCO3 solution and brine. The organic phase was dried over Na2SO4 and the solvent was evaporated. The crude product was purified by column chromatography on silica gel (hexane/EtOAc, 50:50, 0.5% Et3N).

Yield: 330 mg, 0.34 mmol (79%); white foam; Rf  = 0.4 (hexane/EtOAc, 25:75).

1H NMR (300 MHz, DMSO-d 6): δ = 0.04 (s, 3 H, H 3C-Si), 0.05 (s, 3 H, H 3C-Si), 0.82 (s, 9 H, (H 3C)3-C), 1.01–1.06 (d, 12 H, H 3C-CH), 1.82 (s, 3 H, H 3C-CO), 2.22 (s, 3 H, H 3C-CO-N(4)), 2.23–2.70 (t, 2 H, CH 2CN), 3.40–3.53 (m, 6 H, H 2-C(5′), CH 2-P, H3C-CH), 3.71 (s, 6 H, H 3C-O), 4.06 (m, 2 H, H-C(4′), H-C(3′)), 4.18 (m, 1 H, H-C(2′)), 4.38–4.54 (m, 2 H, H 2C-C(5)), 5.77–5.87 (d, 1 H, H-C(1′)), 6.84–6.87 (m, 4 H, H-C(Ar)), 7.20–7.39 (m, 9 H, H-C(Ar)), 8.04 (s, 1 H, H-C(6)), 10.02 (s, 1 H, H-N(4)).

31P NMR (121 MHz, DMSO-d 6): δ = 149.16, 149.45.

MS (ESI): m/z [M + H]+ calcd for C50H69N5O11PSi: 974.45; found: 974.29.

HRMS (ESI, FT-ICR): m/z [M + H]+ calcd for C50H68N5O11PSi: 974.4495; found: 974.4483.


#

5-Acetyloxymethyl-5′-O-(4,4′-dimethoxytrityl)-2′-O-tert-butyldimethylsilyluridine (10)

Compound 3 (450 mg, 0.72 mmol) was dissolved in dry THF (5.6 mL), then dry pyridine (200 μL, 2.5 mmol) was added, followed by AgNO3 (200 mg, 1.2 mmol). The suspension was stirred for 30 min, then TBDMSCl (185 mg, 1.2 mmol) was added. Stirring was continued in the dark for 4 h until the starting material had been consumed as analyzed by TLC. The reaction mixture was filtered through Celite and the Celite bed washed with CH2Cl2 (100 mL). The combined filtrates were evaporated, the residue redissolved in CH2Cl2, and then washed with aq citric acid (2 ×) and sat. NaHCO3 solution. The organic phase was dried over Na2SO4, filtered, and evaporated. The crude product was purified by column chromatography on silica gel (hexane/EtOAc, 80:20–50:50).

Yield: 230 mg, 0.31 mmol (44%); white foam; Rf  = 0.55 (hexane/ EtOAc­, 25:75).

1H NMR (300 MHz, CDCl3): δ = 0.16 (s, 3 H, H 3C-Si), 0.17 (s, 3 H, H 3C-Si), 0.96 (s, 9 H, (H 3C)3-C), 1.87 (s, 3 H, H 3C-CO), 2.70 (d, J = 4.4 Hz, 1 H, HO-C(3′)), 3.40–3.56 (m, 2 H, H 2-C(5′)), 3.82 (s, 6 H, H 3C-O), 4.20 (m, 1 H, H-C(4′)), 4.28 (m, 1 H, H-C(3′)), 4.48 (m, 1 H, H-C(2′)), 4.06–4.32 (m, 2 H, H 2C-C(5)), 6.04 (d, J = 5.0 Hz, 1 H, H-C(1′)), 6.87–6.89 (m, 4 H, H-C(Ar)), 7.28–7.41 (m, 9 H, H-C(Ar)), 7.91 (s, 1 H, H-C(6)), 8.42 (s, 1 H, H-N(3)).

13C NMR (75 MHz, CDCl3): δ = –4.98, –4.59 (2 × CH3-Si), 18.13 (C(CH3)3), 20.79 (COCH3), 25.76 (3 × CH3-C-Si), 55.36 (2 × OCH3), 58.79 (CH2OH), 63.32 (C(5′)), 71.34 (C(3′)), 76.02 (C(2′)), 84.08 (C(4′)), 87.22 (t-C(DMT)), 88.46 (C(1′)), 109.99 (C(5)), 113.50 (C(Ar)), 127.35 (C(Ar)), 128.34 (C(Ar)), 128.34 (C(Ar)), 130.25 (C(Ar)), 135.33 (C(Ar)), 135.46 (C(Ar)), 140.67 (C(6)), 144.23 (C(Ar)), 150.00 (C(2)), 158.91 (C-OCH3(Ar)), 161.89 (C(4)), 170.44 (COCH3).

MS (ESI): m/z [M + Na]+ calcd for C39H48N2O10Si: 755.30; found: 755.44.


#

5-Acetyloxymethyl-5′-O-(4,4′-dimethoxytrityl)-2′-O-tert-butyldimethylsilyluridine 3′-O-(2-Cyanoethyl-N,N-diisopropylphosphoramidite) (11)

Compound 10 (230 mg, 0.30 mmol) was dissolved in dry CH2Cl2 (3.5 mL) under an argon atm. Next, DIPEA (206 μL, 1.2 mmol), 1-meth­ylimidazole (12 μL, 0.15 mmol), and 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (133 μL, 0.60 mmol) were added to the solution using syringes. The reaction was monitored by TLC and the mixture stirred for 5 h at r.t. Then, CH2Cl2 was added and the organic phase extracted with sat. aq NaHCO3 solution and brine. The organic phase was dried over Na2SO4 and the solvent was evaporated. The crude product was purified by column chromatography on silica gel (hexane/EtOAc, 65:35, 0.5% Et3N ).

Yield: 220 mg, 0.24 mmol (80%); white foam; Rf  = 0.5 (hexane/EtOAc, 50:50).

1H NMR (300 MHz, CDCl3): δ = 0.11–0.16 (s, 3 H, H 3C-Si), 0.91–0.93 (s, 9 H, (H 3C)3-C), 1.00–1.20 (d, 12 H, H 3C-CH), 1.82–1.84 (s, 3 H, H 3C-CO), 2.35–2.68 (t, 2 H, CH 2CN), 3.35–3.50 (m, 2 H, H 2-C(5′)), 3.59 (m, 4 H, CH 2-P, H3C-CH)), 3.82 (s, 6 H, H 3C-O), 4.05 (m, 5 H, H-C(4′), H-C(3′), H-C(2′), H 2C-C(5)), 6.01–6.11 (d, 1 H, H-C(1′)), 6.83–6.87 (m, 4 H, H-C(Ar)), 7.28–7.44 (m, 9 H, H-C(Ar)), 7.92–7.93 (s, 1 H, H-C(6)), 8.13 (s, 1 H, H-N(3)).

31P NMR (121 MHz, CDCl3): δ = 149.90, 151.33.

MS (ESI): m/z [M + Na]+ calcd for C48H65N4O11PSi: 955.40; found: 955.49.

HRMS (ESI, FT-ICR): m/z [M + H]+ calcd for C48H65N4O11PSi: 933.4229; found: 933.4224.


#

RNA Solid-Phase Synthesis

Phosphoramidite chemistry was applied for automated RNA strand elongation using 2′-OTOM nucleoside building blocks (GlenResearch) and a polystyrene support (GE Healthcare, Custom Primer SupportTM, 80 μmol/g, PS 200) in combination with the new phosphoramidites 9 and 11. All oligonucleotides were synthesized on an ABI 392 Nucleic Acid Synthesizer following standard methods: detritylation (2.0 min) with dichloroacetic acid/1,2-dichloroethane (4:96), coupling (6.0 min) with phosphoramidites/MeCN (0.1 M × 130 μL) and 5-benzylthio-1H-tetrazole/MeCN (0.3 M × 360 μL), capping (2 × 20 sec, Cap A/Cap B = 1:1) with Cap A: DMAP in MeCN (0.5 M) and Cap B: Ac2O/sym-collidine/MeCN (2:3:5), oxidation (1.0 min) with I2 (20 mM) in THF/pyridine/H2O (35:10:5). The solutions of amidites and tetrazole, and MeCN were dried over activated molecular sieves (3 Å) overnight.


#

Deprotection of 5-Hydroxymethylpyrimidine-Modified RNA

The solid support was removed and treated with MeNH2 in EtOH (33%, 0.65 mL) and MeNH2 in H2O (40%, 0.65 mL) for 4 h at r.t. for short sequences (<10 nt), and for 4 h at 35 °C for longer sequences (>10 nt). Alternatively, the solid support was treated with MeNH2 in H2O (40%, 0.65 mL) and NH3 in H2O (28%, 0.65 mL) for 30 min at 65 °C. The supernatant was removed and the solid support was washed three times with EtOH/H2O (1:1). The supernatant and the washings were combined with the deprotection solution of the residue and the whole mixture was evaporated to dryness. To remove the 2′-silyl protecting groups, the resulting residue was treated with tetrabutylammonium fluoride trihydrate (TBAF·3H2O) in THF (1 M, 1 mL) at 37 °C overnight. The reaction was quenched by the addition of triethylammonium acetate (TEAA) (1 M, pH 7.4, 1 mL). The volume of the solution was reduced and the solution was desalted with a size-exclusion column (GE Healthcare, HiPrep™ 26/10 Desalting; 2.6 × 10 cm, Sephadex G25) eluting with H2O, and the collected fraction was evaporated to dryness and dissolved in H2O (1 mL). Analysis of the crude RNA after deprotection was performed by anion-exchange chromatography on a Dionex DNAPac® PA-100 column (4 mm × 250 mm) at 80 °C. Flow rate: 1 mL/min, eluent A: 25 mM Tris·HCl (pH 8.0), 6 M urea; eluent B: tris·HCl (25 mM) (pH 8.0), NaClO4 (0.5 M), urea (6 M); gradient: 0–60% B in A within 45 min and UV detection at 260 nm.


#

Purification of 5-Hydroxymethylpyrimidine-Modified RNA

Crude RNA products were purified on a semipreparative Dionex DNAPac® PA-100 column (9 mm × 250 mm) at 80 °C with a flow rate of 2 mL/min. Fractions containing RNA were loaded on a C18 SepPak Plus® cartridge (Waters/Millipore), washed with 0.1–0.15 M (Et3NH)+HCO3– and H2O, and eluted with H2O/MeCN (1:1). RNA-containing fractions were evaporated to dryness and dissolved in H2O (1 mL). Analysis of the quality of purified RNA was performed by anion-exchange chromatography under the same conditions as utilized for crude RNA; the molecular weight was confirmed by LC–ESI mass spectrometry. Yield determination was performed by UV photometrical analysis of oligonucleotide solutions.


#

Mass Spectrometry of 5-Hydroxymethylpyrimidine-Modified RNA

All experiments were performed on a Finnigan LCQ Advantage MAX ion trap instrument connected to an Amersham Ettan micro LC system. RNA sequences were analyzed in the negative-ion mode with a potential of –4 kV applied to the spray needle. LC: Sample (200 pmol RNA dissolved in 30 μL of 20 mM EDTA solution; average injection volume: 30 μL), column (Waters XTerra®MS, C18 2.5 μm; 2.1 × 50 mm) at 21 °C; flow rate: 30 μL/min; eluent A: Et3N (8.6 mM), 1,1,1,3,3,3-hexafluoroisopropanol (100 mM) in H2O (pH 8.0); eluent B: MeOH; gradient: 0–100% B in A within 30 min; UV detection at 254 nm.


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#

Acknowledgment

This research was supported by the Austrian Science Fund FWF (I1040, P27947). We thank Heidelinde Glasner and Kathrin Breuker (University of Innsbruck) for FT-ICR high-resolution mass spectra, and Thomas Amort and Alexandra Lusser (Biocenter, Medical University of Innsbruck) for stimulating discussions on RNA epigenetics and critical reading of the manuscript.

Supporting Information

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Figure 1 Different protection patterns of 5-hydroxymethyl-2′-deoxycytidine phosphoramidites for the solid-phase synthesis of 5hm(dC)-containing DNA that have been reported in the literature
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Scheme 1 Synthesis of 5hm(rC) phosphoramidite 9. Reaction conditions: (a) TFA (0.0002 equiv), AcOH, reflux, 40 min, 62%; (b) DMT-Cl (1.2 equiv), DMAP (0.025 equiv), pyridine, 5 h, r.t., 65%; (c) TBDMSCl (3.0 equiv), imidazole (6.0 equiv), DMF, r.t., 5 h, 80%; (d) 2,4,6-triisopropylbenzenesulfonyl chloride (1.5 equiv), Et3N (10.0 equiv), DMAP (0.12 equiv), CH2Cl2, r.t., 1 h, no purification; (e) aq NH3 (32%), THF, r.t., 3 h, 44% over 2 steps; (f) Ac2O (2.5 equiv), pyridine, 0 °C to r.t., 2 h, 76%; (g) 1 M TBAF/0.5 M AcOH, THF, r.t., 3 h, 75%; (h) AgNO3 (1.6 equiv), TBDMSCl (1.6 equiv), pyridine (3.5 equiv), THF, r.t., 4 h, 43%; (i) CEP-Cl (2.0 equiv), DIPEA (4.0 equiv), 1-methylimidazole (0.5 equiv), CH2Cl2, r.t., 5 h, 79%.
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Scheme 2 Synthesis of 5hm(rU) phosphoramidite 11. Reaction conditions: (a) AgNO3 (1.6 equiv), TBDMSCl (1.6 equiv), pyridine (3.5 equiv), THF, r.t., 4 h, 44%; (b) CEP-Cl (2.0 equiv), DIPEA (4.0 equiv), 1-methylimidazole (0.5 equiv), CH2Cl2, r.t., 5 h, 80%.
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Figure 2 Characterization of 5hm(rC)- and 5hm(rU)-modified RNA. Anion-exchange HPLC traces (top) of: (A) 8 nt RNA, (B) 15 nt RNA, and (C) 15 nt RNA, and the corresponding LC–ESI mass spectra (bottom). HPLC conditions: Dionex DNAPac column (4 × 250 mm), 80 °C, 1 mL min–1, 0–60% buffer B in buffer A within 45 min; buffer A: Tris-HCl (25 mM), urea (6 M), pH 8.0; buffer B: Tris-HCl (25 mM), urea (6 M), NaClO4 (0.5 M), pH 8.0. See the experimental for LC–ESI MS conditions